Manipulation of organisms or in vitro cultures to alter and/or improve phenotypic characteristic often requires the modulation of gene expression. For example, the stress tolerance of a plant can be improved through modifying the expression of genes involved in signal transduction pathways related to various stress responses. One way to achieve this goal is to genetically engineer the organisms or in vitro cultures, an approach that is costly and time consuming. An alternative approach is to identify chemical compounds that can be applied to these organisms, e.g., plants, mammals, yeast, Drosophila, C. elegans, or bacteria etc., or their in vitro cultures to obtain the desired phenotypes. Currently, chemicals are screened through reporter constructs in which a reporter polynucleotide is directly fused to a promoter sequence that is capable of being recognized by a transcriptional regulatory protein, i.e., proteins that can regulate the signaling pathways that contribute to the development of a desired trait. However, this conventional approach has limited sensitivity that leads to inefficient compound screening and/or requires significant effort to identify reporter cell lines or organisms suitable for the screen, i.e., reporter cell lines or organisms in which the function or expression of the transcriptional regulatory protein can be altered directly or indirectly in response to a compound treatment so that the amount of the resulting reporter molecule is readily discernible from the controls.
The present specification provides, inter alia, novel vectors, cell lines, and methods useful for modulating gene expression, identifying and analyzing regulatory sequences, and discovering new targets and reagents for improving plant performance or therapeutic intervention in human disease. The reporter constructs of the specification contain arrangements of additional genetic elements that can optimize the signal-to-noise ratio of the conventional promoter-reporter assay, minimize the efforts on characterization and development of reporter lines, and thus improve the sensitivity and the efficiency of the screens. These novel constructs and methods can also be used in a high-throughput format to identify agents that can be rapidly deployed induce modified gene expression and/or desired phenotypic alterations in organisms, for example, compounds can be applied to plants through a spray or via irrigation.
Examples of how to employ these reporter constructs and transgenic cells and organisms to identify useful chemical compounds are provided. Other aspects and embodiments of the specification are described below or can be derived from the teachings of this disclosure as a whole.